This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Glandular lesions are often problematic for diagnostic cervical pathology. The survival
of patients with adenocarcinoma is significantly poorer than that of patient with
squamous cell carcinoma. One reason for this increased risk is the aggressive invasiveness
of adenocarcinoma. Therefore additional biomarkers, to supplement morphological diagnosis
of adenocarcinoma, are necessary. We have assessed the diagnostic utility of Laminin-5
(Laminin γ2 chain): Lam-5 in the diagnosis of the invasiveness of cervical adenocarcinoma
and related glandular lesions.

Methods

Lam-5 immunohistochemistry was performed on archival specimens from 8 patients with
uterine leiomyoma as a negative control group, 6 patients with endocervical gland
hyperplasia, 6 patients with adenocarcinoma in situ, 6 patients with microinvasive
adenocarcinoma and 24 patients with invasive adenocarcinoma.

Results

The expression of Lam-5 was not detected in normal mucosa, but was seen along the
basement membrane in endocervical gland hyperplasia and adenocarcinoma in situ and
was observed in the cytoplasm of tumor cells in microinvasive and invasive adenocarcinoma.

Conclusion

We conclude that Lam-5 is a useful biomarker in the evaluation of invasiveness in
cervical adenocarcinoma.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7316562925827381

Keywords:

Background

Although adenocarcinoma (AC) is rare, relative to squamous cell carcinoma (SCC), among
uterine cervical cancers, the number of cases has increased in recent years, particularly
in young women [1]. Like SCC, invasive AC is associated with high-risk human papillomavirus infection
and arises from non-invasive precursors, namely cervical glandular intraepithelial
neoplasia/adenocarcinoma in situ [2]. The distinction between AC and SCC is not only of academic interest since these
glandular lesions present many problems for diagnostic cervical pathology. Many different
types of benign glandular lesions of the endocervix increase the potential for cervical
neoplasia [3]. However, the distinguishing features of AC and SCC have practical therapeutic and
prognostic implications, since the survival of patients with AC is significantly poorer
than that for patients with SCC. The high invasiveness of tumor cells is the main
reason for poor prognosis in AC. Many factors affect the invasiveness of tumors. A
wide variety of molecular markers have been evaluated as diagnostic tools in the identification
of high-risk precursors of SCC and AC and the association with cell adhesion molecules
has attracted attention recently. These molecules affect the protein constitution
of the basement membrane and play an important role in invasiveness.

Laminin-5 (Laminin γ2 chain): Lam-5 consists of extracellular proteins and is among
the components of the basement membrane. The major functions of Lam-5 include binding
of epithelial cells to the basement membrane through the formation of hemidesmosomes
[4] and the migration of epithelial cells during wound repair [5,6]. In addition, it has been reported that there is a correlation between its expression
and tumor progression in various kinds of malignant tumors [7-20]. According to some reports, Lam-5 is found in front-line invasive tumor cells at
the epithelial-stromal interface and plays an important role in cancer cell invasion
[9,11,12,17-23]. Accumulating data indicates that Lam-5 expression can serve as a marker for invasiveness
in carcinoma cells from different tissues [18,19,23]. In uterine cervical cancers, recent data suggests that expression of Lam-5 could
be a useful marker in the detection of invasive squamous cell carcinoma [20]. A more interesting finding, perhaps is that Lam-5 seems to be expressed from the
earliest stages, particularly in microinvasive lesions.

Lam-5 may therefore become a very useful biomarker of early invasion in the AC of
the uterine cervix. The aim of the present study was to assess whether Lam-5 can be
used for evaluation of tumor cell invasiveness in cervical AC and other precancerous
lesions. We also investigated whether Lam-5 can be used as a marker of invasiveness
in neoplastic lesions.

Methods

Cases

Each of our 50 cases were registered at the Department of Surgical and Molecular Pathology,
Dokkyo University School of Medicine and the Department of Pathology, Ibaraki Prefectural
Central Hospital and were available for clinicopathological and immunohistochemical
analysis. The patients were staged clinically according to the criteria of the International
Federation of Gynecology and Obstetrics: FIGO. Of the 42 tumors, 6 were glandular
hyperplasia, 6 were adenocarcinoma in situ (TMN Stage 0), 6 were microinvasive adenocarcinoma
(Stage Ia) and 24 were invasive carcinoma (Ib were 16 cases, II were 5 cases and III
were 3 cases).

Immunohistochemistry

The control specimens were normal tissues from 8 cases of hysterectomy with diagnosis
based on uterine leiomyoma. We selected routinely-processed, formalin-fixed, paraffin-embedded
tissue blocks and prepared 5-micrometer serial sections from the cut surface of the
blocks. Mouse monoclonal antibody (clone D4B45, Chemicon 1:100) was used for the present
immunohistochemical study. Immunoperoxidase reactions were performed using a Venta
View automated immunostainer according to the manufacturer’s instructions. All cases
were reviewed by two investigators (JI and TF), and were provided with a consensus
on the pathological diagnoses and the assessment of immunoreactivity.

Results

No expression of Lam-5 was seen in normal endocervical glands and surrounding stroma
(Figure 1A). Two of 6 cases (33%) of endocervical gland hyperplasia showed linear or focal
basement membrane expression of Lam-5 (Figure 1B). Five of 6 (83%) cases of ’adenocarcinoma in situ’ showed continuous and linear
Lam-5 expression along the basement membrane (Figure 1C). All of the 6 cases (100%) of microinvasive adenocarcinoma produced a positive
reaction, with expression mainly present at the invading front. Four of these cases
evidenced cytoplasmic expression with the remainder linearly expressed along the basement
membrane. Cytoplasmic expression was specifically seen in budding or dissociated cells
from tumor nests. Eighteen of 24 cases (75%) of invasive adenocarcinoma revealed cytoplasmic
expression (Figure 1D). Cytoplasmic expression was diffuse in most tumor cells and scattered in the stroma.
Linear expression along the basement membrane surrounding tumor cells was also observed
in 11 of these cases. The immunohistochemical findings are summarized in Table 1.

Figure 1.Immunohistochemical findings for Laminin-5 in the normal mucosa and the lesions of
uterine cervix.A. Normal mucosa: No reaction in the endocervical glands. B. Endocervical gland hyperplasia and C. Adenocarcinoma in situ: Continuous and linear expression along basement membrane.
D. Invasive adenocarcinoma: The diffuse immunoreactivity in most of tumor cells is
scattered in the stroma.

Discussion

Laminins are a family of basement membrane proteins, which associate with cell differentiation
proteins, adhesion and migration proteins, as well as being structural components
themselves [24-26]. Lam-5 is a recently identified laminin isoform and is a disulfide-linked heterotrimer
with precursor subunits of 200-, 155-, and 140-kDa. These precursors are rapidly processed
by cleavage of the 200- and 155-kDa subchains into 165- and 105-kDd polypeptides,
respectively [27]. Lam-5 is known to be expressed in squamous cell carcinoma, various adenocarcinomas
and in organs such as the esophagus, cervix, breast, colon and pancreas [7-20]. An earlier study proposed that binding of the ligand to urinary-type plasminogen
activator receptor: uPAR promotes cancer cell invasion by activation of plasminogen,
leading to the degradation of extracellular matrix [28]. The coexpression of Lam-5 and uPAR suggested Lam-5 may be useful as a marker of
invasion in some human cancers [18,23]. According to certain studies, Lam-5 is found at the invasive front, along the epithelial-stromal
interface and plays an important role in cancer cell invasion [9,11,12,17-23]. Sordat et al. [23] and Pyke et al. [17,18] observed budding cancer cells linked with the accumulation of Lam-5 in the cytoplasm.
In contrast, extracellular Lam-5 expression has been reported in gastric cancer and
in basement membranes surrounding cancer cells [11].

Lam-5 is expressed in hyperplastic tissue and most tumors, but is not expressed in
normal tissue. Although the expression of Lam-5 was shown in a few glands with endocervical
hyperplasia, these cells might have already acquired tumor characteristics without
morphological changes i.e. precursor cells derived from endocervical glands that expressed
Lam-5 might acquire tumor characteristics.

Based on our results, we suggest that Lam-5 expression might be an early event during
neoplastic progression towards AC. According to our hypothesis, the upregulation of
expression of Lam-5 might be related to tumor progression, e.g. the adenoma-noninvasive
carcinoma-invasive carcinoma sequence seen in colon carcinogenesis. It seems clear
from the literature that structural changes in Lam-5 are relatively early events during
the development of invasiveness. On the other hand, the frequency of Lam-5 expression
decreased, to 75% in invasive AC although it is 100% in microinvasive AC. This interpretation
is difficult, but Lam-5 may be expressed at an early stage of the invasion. Furthermore
the expression of Lam-5 might be attenuated in the later phases when tumor cells are
more extensively invasive. Although the detailed molecular carcinogenic mechanism
is unknown, it does seem to be related to Lam-5 expression and the acquirement of
invasiveness. Several investigators have shown that the Lam-5 chain is expressed in
epithelial cells at the invasive front of malignant tumors [9,11,12,17-23]. Other investigators also describe how Lam-5 could become a useful biomarker for
the early detection of invasive tumor cells [20,29,30].

Cervical cancer is the second most common malignancy in women worldwide [31]. Of the various histologic subtypes, SCC is by far the most common form while AC
is relatively rare. In recent years, epidemiologic studies have shown an increasing
incidence of AC worldwide [32], especially in younger patients [1,33]. AC is a highly aggressive gynecologic malignancy. Over the last several decades,
the relative proportion of AC to SCC of the uterine cervix has been increasing with
several studies reporting an increase in the absolute number of AC cases [34]. In uterine cervical neoplasia, there are many etiologies that explain why the AC
patient has a poorer prognosis than that of SCC [35]. One is the high tendency for invasiveness of adenocarcinoma cells. It is often hard
to diagnose by colposcopy, cytodiagnosis and punch biopsy, since AC tends to grow
endophytically. With regard to diagnosis, differentiation between adenocarcinoma in
situ (FIGO 0), microinvasive (Ia), and invasive adenocarcinoma (Ib) is an important
and difficult question to answer. The treatment strategy for patients is different
for adenocarcinoma in situ, microinvasive adenocarcinoma and invasive adenocarcinoma.
The patient will receive disease-specific treatments e.g. conization in adenocarcinoma
in situ, simple hysterectomy in microinvasive adenocarcinoma and radical hysterectomy
with additional lymphadenectomy in invasive adenocarcinoma. It is often difficult
to make this decision preoperatively. We conclude in this article that the immunohistochemistry
of Lam-5 is effective as a means to solve this problem. Specifically, when cells expressing
Lam-5 were observed in the lesion by biopsy, we are able to surmise that those cells
are invasive. Therefore a simple hysterectomy and/or more extensive surgery is indicated
for these patients. In addition, even if the lesion was non-invasive in CIS morphologically,
these tumor cells might have acquired invasiveness when the cells express Lam-5. In
such a case, intensive follow-up care is required after treatment by conization. Thus,
a sensitive diagnostic procedure is a prerequisite for appropriate therapy.

Conclusion

It is only cases of microinvasive or invasive adenocarcinoma that have shown cytoplasmic
immunoreactivity to Lam-5. These findings are similar to other reports that have compared
immunoreactivity in different tumors, particularly cervical squamous neoplasia. Our
results indicate that immunohistochemical expression of Lam-5 is a useful marker for
detecting invasive tumor cells in AC. We can evaluate invasion in AC more precisely
using Lam-5 immunohistochemistry as an adjunct to morphological examination. This
method could be useful in the histopathological diagnosis of cervical cancer.

Abbreviations

Competing interests

No financial and non-financial competing interests to declare in relation to this
manuscript.

Authors' contributions

JI was involved in the design of the study and immunohistochemical analysis, and drafted
the manuscript. YU conceived the study, was involved in the design and immunohistochemical
analysis, and edited the manuscript for intellectual content. KI, ST, TI and TF were
involved in the design of the study and pathological diagnosis. All authors read and
approved the final manuscript.

Sonnenberg A, de Melker AA, Martinez de Velasco AM, Janssen H, Calafat J, Niessen CM: Formation of hemidesmosomes in cells of a transformed murine mammary tumor cell line
and mechanisms involved in adherence of these cells to laminin and kalinin.

Gagnoux-Palacios L, Allegra M, Spirito F, Pommeret O, Romero C, Ortonne JP, Meneguzzi G: The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation
of laminin 5 into the extracellular matrix and in cell adhesion.

Alfsen GC, Thoresen SO, Kristensen GB, Skovlund E, Abeler VM: Histopathologic subtyping of cervical adenocarcinoma reveals increasing incidence
rates of endometrioid tumors in all age groups: a population based study with review
of all nonsquamous cervical carcinomas in Norway from 1966 to 1970, 1976 to 1980,
and 1986 to 1990.